The search for novel antimicrobial agents is intensifying, in response to both the threat of microbial pathogens in bioterrorism and the increasing development of drug resistance to antibiotic therapeutics currently in use. Antimicrobial peptides are essential host defense molecules found in a wide variety of species and are promising antibacterial therapeutic candidates (Zasloff, M. (2002) Nature, 415:389-395; McPhee et al. (2005) J. Pept. Sci., 11:677-687). Several hundreds of antimicrobial peptides have been identified in a variety of life forms ranging from bacteria, fungi, plants, amphibians, to mammals, including humans (Mygind et al. (2005) Nature, 437:975-80; Brahmachary et al. (2004) Nucleic Acids Res., 32:D586-589; Wang et al. (2004) Nucleic Acids Res., 32:D590-D592). In mammals, cathelicidins and defensins are the two major types of host defense peptides (Zanetti, M. J. (2004) Leukoc. Biol., 75:39-48). Defensins usually contain three pairs of disulfide bonds that stabilize the protein fold. Cathelicidins, however, are rather variable in both sequence and structure, although their precursor proteins share a common N-terminal “cathelin” domain. Cathelicidins are classified into three groups. The first group of cathelicidin peptides contains 12-18 residues with beta-hairpin structures stabilized by one or two disulfide bonds. This group also includes a 13-residue linear peptide with a high content of tryptophans. The second group contains 23-37 residues and has the potential to form a helical structure. The peptides in the third group such as PR-39 are rich in prolines with 39-80 residues (for a review, see Zanetti, M. J. (2004) Leukoc. Biol., 75:39-48).
LL-37 is the only human cathelicidin identified to date. LL-37 is 37 amino acids in length and has two leucines at its N-terminus. It has been detected in a variety of cells such as B cells, monocytes, mast cells, and immature neutrophils (Sorensen et al. (1997) Blood, 90:2796-2803; Agerberth et al. (2000) Blood, 96:3086-3093; Di Nardo et al. (2003) J. Immunol., 170:2274-2278). Several lines of evidence support the significance of this human peptide in host defense. First, the precursor gene of LL-37 (hCAP-18) is up-regulated in skin in response to cutaneous infection as well as in inflammatory skin disorders such as psoriasis (Dorschner et al. (2001) Invest. Dermatol., 117:91-97; Frohm et al. (1997) J. Biol. Chem., 272:15258-15263). Second, LL-37 deficiency in neutrophils correlates with the occurrence of chronic periodontal diseases in patients with morbus Kostmann (Putsep et al. (2002) Lancet, 360:1144-1149). Third, gene knockout of the CRAMP cathelicidin in mice increases their susceptibility to skin infection (Nizet et al. (2001) Nature, 414:454-457). Fourth, expression of additional cathelicidins by gene transfer protects against skin infection by bacteria (Lee et al. (2005) Proc. Natl. Acad. Sci. U.S.A., 102:3750-3755). In addition to its antibacterial effects, human LL-37 appears to play an important role in angiogenesis, chemotaxis, and signal transduction as well (Zanetti, M. J. (2004) Leukoc. Biol., 75:39-48; Bowdish et al. (2004) J. Immunol., 172:3758-3765; Tjabring a et al. (2003) J. Immunol., 171:6690-6696). Further, after secretion onto the skin surface, human LL-37 in sweat can be cleaved (e.g., after residue F6 or R7) into more active antibacterial and antifungal fragments with a reduced toxicity to erythrocytes (Murakami et al. (2004) J Immunol., 172:3070-3077). Further, these shorter forms of LL-37 lost their capability of stimulating a host response possessed by the full-length peptide. This important observation indicates that a potent antibacterial region can be identified within LL-37 as a peptide template for therapeutic use.
According to previous circular dichroism (CD) studies, LL-37 forms helical structures upon increasing peptide concentration, anions, pH, detergents, and lipids (Johansson et al. (1998) J. Biol. Chem., 273:3718-24; Oren et al. (1999) Biochem. J., 341:501-13). The helicity of the peptide was found to correlate with antibacterial activity. Recent solid-state NMR, differential scanning calorimetry, and biochemical analysis substantiated the interactions of LL-37 with lipid bilayers (Johansson et al. (1998) J. Biol. Chem., 273:3718-24; Oren et al. (1999) Biochem. J., 341:501-13; Henzler-Wildman et al. (2004) Biochemistry, 43:8459-69; Henzler-Wildman et al. (2003) Biochemistry, 42:6545-58). Three-dimensional structure is essential for understanding the mechanism of action of the peptide. However, no three-dimensional structure has been reported for LL-37.